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Original Article

Anti-microbial efficacy of green tea and chlorhexidine


mouth rinses against Streptococcus mutans,
Lactobacilli spp. and Candida albicans in children
with severe early childhood caries: A randomized
clinical study
Ann Thomas, Sneha R Thakur, Sowmya B Shetty
Department of Pedodontics and Preventive Dentistry, A. J. Institute of Dental Sciences, Mangalore, Karnataka, India

ABSTRACT Address for correspondence:


Dr. Sneha R Thakur,
Introduction: Green tea is a beverage which is consumed
Department of Pedodontics and Preventive Dentistry, A. J.
worldwide and is reported to have anti-cariogenic effect. Institute of Dental Sciences, Kuntikana, Mangalore - 575 004,
So, if it was as effective as chlorhexidine (CHX) mouth Karnataka, India.
rinse against cariogenic microbes it could be considered E-mail: snehathakur68@gmail.com
a natural, economical alternative. The purpose of this
study was to evaluate and compare the anti-microbial
efficacy of 0.5% green tea and 0.2% CHX mouth rinses Access this article online
against Streptococcus Mutans, Lactobacilli spp. and Quick response code Website:
Candida Albicans. Materials and Methods: 30 children www.jisppd.com
aged 4-6 years with S-ECC (based ondefs score) were DOI:
selected. Children were divided randomly into 2 equal 10.4103/0970-4388.175518
groups and were asked to rinse with the prescribed
PMID:
mouth rinse once daily for 2 weeks after breakfast under
26838151
supervision. A base-line and post rinsing non-stimulated
whole salivary sample (2 ml) was collected and tested
for the number of colony forming units. The data was and Romans. As early as 40-90 A. D, Pedanius
statistically analyzed using SPSS v16.0 software with Dioscorides, a Greek physician suggested a concoction
one-way ANOVA and Tukey’sPOSTHOC test. Results: of olive juice, pomegranate fillings, wine, and gum
A statistically significant fall in colony count was found myrrh to treat halitosis, while Hippocrates is known
with both the mouth rinses in Streptococcus Mutans (P to have advocated mouth rinsing with a mixture of
< 0.001, P < 0.001) and lactobacilli (P < 0.001, P < 0.001) alum, salt, and vinegar in the ancient times.[1] Mouth
but not against Candida albicans (P = 0.264, P = 0.264). rinses are used generally for their analgesic, anti-
Against Streptococcus Mutans, green tea mouth rinse inflammatory, anti-microbial, and anti-cariogenic
was found to be significantly better than CHX mouth
This is an open access article distributed under the terms of the Creative
rinse (P = 0.005). Against lactobacilli spp, CHX mouth
Commons Attribution-NonCommercial-ShareAlike 3.0 License, which
rinse was significantly better than green tea mouth rinse
allows others to remix, tweak, and build upon the work non-commercially,
(P < 0.001). Conclusion: Green tea mouth rinse can be
as long as the author is credited and the new creations are licensed under
considered safe, economical and used without much the identical terms.
concern. However, further studies are recommended.
For reprints contact: reprints@medknow.com
KEYWORDS: Herbal mouth wash, severe early
childhood caries, home oral hygiene, caries prevention How to cite this article: Thomas A, Thakur SR, Shetty SB.
Anti-microbial efficacy of green tea and chlorhexidine mouth
rinses against Streptococcus mutans, Lactobacilli spp. and
Introduction Candida albicans in children with severe early childhood
caries: A randomized clinical study. J Indian Soc Pedod Prev
The use of mouth rinses as an aide to other oral hygiene
Dent 2016;34:65-70.
measures was first propagated by ancient Egyptians

© 2016 Journal of Indian Society of Pedodontics and Preventive Dentistry | Published by Wolters Kluwer - Medknow 65
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Thomas, et al.: Comparing anti-microbial efficacy of green tea and chlorhexidine mouth rinses on cariogenic microbes

activities. Currently, a wide range of mouth rinses such Worldwide, tea is consumed as a beverage for centuries
as chlorhexidine (CHX), sodium fluoride, and essential and has shown many health benefits. It is also a
oils are available commercially. The American Dental condiment available in most homes. Some reports in
Association (ADA) recommends that mouth rinses the Japanese literature have quoted that “those who
should be effective at modifying the micro-biota by continuously drink a large amount of green tea have
selectively eliminating pathogens without negatively less tooth decay”[10] and “drinking green tea makes
affecting the normal commensals of oral cavity.[2] the mouth clean.”[11] Green tea is reported to be very
Evidence illustrates that the long-term twice daily rich in fluoride and catechin, a bioactive component,
use of anti-plaque and anti-gingivitis mouth rinses, which exerts an anti-cariogenic effect by inhibiting
namely 0.12% CHX gluconate and essential oils with the proliferation of the streptococcal agent, interfering
methyl salicylate which are approved by the Council with the process of bacterial adhesion to tooth enamel
on Dental Therapeutics of the ADA, do not have any and also by inhibiting glucosyltransferase.[12-14] It also
negative effect on the oral microbial flora.[2] Studies mediates protective salivary components such as
have also demonstrated that the use of mouth rinses secretory immunoglobulins, lysozymes, lactoferrin,
in children provided a significant reduction in the oral peroxidases histatins, mucins, or others, thus
decayed extracted filled surface (defs) index.[2,3] exhibiting an indirect anti-cariogenic effect.[15]
Pharmacokinetic studies have shown that after rinsing
The most common plaque-mediated disease in children with tea, catechins can be found in the saliva for up
is dental caries, which is also one of the most common to 60 min and that the enzymatic breakdown of starch
childhood diseases. It is a preventable, localized on food particles trapped in the mouth is noticeably
infectious, multi-factorial disease resulting from the reduced.[14] Studies on the anti-cariogenic effect of green
interaction among host, diet, and microflora on the tea polyphenols have revealed that these compounds
tooth surface over a period of time, resulting in localized have a high inhibitory effect against S. mutans and
de-mineralization of hard tissues. The most implicated the acids produced by it.[14] The cariogenic activity of
bacteria in its etiology are Streptococcus mutans for its catechins present in green tea was found to be related
initiation and Lactobacillus acidophilus for its progression. to its role in the depletion of thiol group which in turn
Recently, a number of reports in the scientific literature exerted bactericidal effect.[14]
show an association between Candida albicans, a fungi,
which is an opportunistic pathogen and a normal To our knowledge, there is a paucity of reports on
commensal of the oral cavity and progression of dental comparing the anti-microbial efficacy of green tea
caries.[4,5] Dental caries in children below 6 years is mouth rinse to that of CHX, the “gold standard,” and
called early childhood caries (ECC), and its severe also no reports regarding its antifungal effect against
debilitating form is called severe-ECC (S-ECC). C. albicans, which is recently being implicated in the
etiology of caries. Thus, the main purpose of this study
Of the commercially available mouth rinses, CHX was to evaluate and compare the anti-microbial efficacy
mouth rinse is widely used and is considered to be of green tea (0.5%) mouth rinse to that of CHX (0.2%)
the “gold standard” due to its broad spectrum anti- against S. mutans, Lactobacilli spp., and C. albicans.
microbial activity. CHX de-stabilizes the bacterial
cell wall and interferes with osmosis. In addition, the Materials and Methods
bacterial uptake of CHX is very rapid, which facilitates
rupture of the cell wall and finally the cytoplasmic A randomized, double-blind, active, controlled clinical
membrane causing cell death.[6-8] However, Lindhe trial was conducted at a local preschool between
et al., reported a number of local side-effects with November 2013 and February 2014. The study protocol
CHX use such as discoloration of teeth, altered taste was reviewed and approved by the Ethical Committee
sensation, mucosal irritation, parotid swelling, and of the Institution and the study was in compliance
enhanced supra-gingival calculus formation due to with The Code of Ethics of the World Medication
precipitation of salivary proteins and organic salts, (Declaration of Helsinki) for experiments involving
which limits its use as a therapeutic agent.[9] human subjects. An informed consent was obtained
from authorities of the preschool and the parents of the
The ease of availability and affordability of commercial subjects before the onset of the study.
mouth rinses is a major hurdle faced by the low
socioeconomic communities. Thus, to achieve better To conduct the study, 30 children aged 4-6 years were
oral health for masses, there is always a constant quest randomly selected from the preschool. Physically
to identify and promote newer, economical products fit children with S-ECC as per the definition given
that can be easily procured even by the people living in by the American Academy of Pediatric Dentistry[16]
remote areas where advanced healthcare facilities are were included in the study. Children who were
inadequate. Identification of an effective mouth rinse unable to expectorate completely or brush their
formulation containing a commonly used condiment teeth on their own, had positive history of taking
would prove highly beneficial and currently a number antibiotics 3 months prior to and during the study
of them are reported in the scientific literature. period, undergoing orthodontic treatment or with an

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Thomas, et al.: Comparing anti-microbial efficacy of green tea and chlorhexidine mouth rinses on cariogenic microbes

intraoral prosthesis, and had any intraoral pathology respectively (HiMedia Laboratories, Mumbai). All the
were medically compromised or for whom parental saliva samples were serially diluted using physiological
consent was not given were also not included saline to obtain −3 concentration. A volume of 0.1 ml
in the study. saliva sample was spread on the selective agar plates
using a sterile glass spreader. The plates were incubated
The defs of the children was recorded using visible for 48 h at 37°C in the incubation chamber (ROTEK)
light, mouth mirror, and community periodontal to obtain maximum growth of microbial colonies.
index probe. The sum total of defs was considered The CFU were identified by morphology, size, and
and based on the caries experience, children were color, and counting was done with a handheld digital
selected for the study. All the study participants were colony counter (HiMedia, Mumbai). The colony count
given a tube of nonfluoridated tooth paste and a tooth was expressed as the number of CFUs per millimeter
brush. The brushing and mouth rinsing technique (CFU/ml) of saliva. Semi-quantification of the number
was demonstrated to all and were advised to brush of colonies was done by multiplying the actual colony
twice daily. The participants were randomly divided count with 1 × 103 to adjust for the dilution factor. The
into two groups of 15 each by lottery method, i.e., colony counting was done by the same observer under
they were asked to pick up chits with the name of the same conditions and at the same time of the day to
the mouth rinse written on them. The children were avoid the intraobserver variability in the results.
given prenumbered identical mouth rinse bottles and
advised to rinse the mouth for 1 min using 5 ml of the Evaluation of acceptability
respective mouth rinse daily for 2 weeks in the school. After completion of the study, the study participants
Rinsing was carried out under the supervision of the were given a self-administered, close-ended
investigator for six working days of the week and on dichotomous questionnaire regarding the acceptability
Sundays under parental supervision. of mouth rinse prescribed to each of them. The
questionnaire consisted of three questions with two
Preparation of mouth rinses mutually exclusive options (YES/NO) to answer.
Green tea mouth rinse was custom prepared by the The questions were based on the acceptability of the
pharmacist. To prepare green tea mouth rinse, dried mouth rinses in terms of flavor, smell, and willingness
green tea leaves (obtained by open air drying) were to continue using the mouth rinse. A paper and pencil
grounded to a desirable size using an electrical mill, method was used to administer the questionnaire. With
and then extracted by percolation using distilled the help of the caretaker, the participants’ response
water as solvent. Green tea which is rich in phenolic was elicited and the questionnaire was completed.
compounds (6%) was diluted to obtain a concentration
of 0.5% phenolic compound using double distilled Outcome measures and statistical methods
water. Authorized additive, peppermint flavor The study had a single end point at 2 weeks. The data were
(1 g/l), and sodium saccharine (1 g/l), a sweetening statistically analyzed using one-way variance ANOVA
agent, were used to formulate the mouth rinse.[17] and Tukey’s post-hoc honestly significant difference
Commercially available CHX (0.2%) mouth rinse (HSD) test in SPSS Software 16.0 (SPSS Inc., Chicago).
(CLOHEX, Dr. Reddy’s) was used in this study. The results were considered statistically significant at
0.05 probability level. One-way variance ANOVA test
Saliva samples was used to compare the mean of differential colony
A volume of 2 ml saliva samples were collected counts in the two mouth rinse groups and to assess
prior to the commencement of mouth rinsing, i.e., at the anti-microbial efficacy of green tea mouth rinse,
baseline and after 2 weeks rinsing, i.e., postrinsing. which was the primary outcome of the study. Tukey’s
Un-stimulated whole saliva samples were collected post-hoc HSD test was used for comparative analysis of
by asking the children to drool into a sterile container two mouth rinse groups and to measure the secondary
for 3-5 min, sitting in an upright position in a well outcome, i.e., whether the newly formulated green tea
lit room with good ventilation. They were collected mouth rinse was better than CHX mouth rinse.
in the morning between 10.00 and 11.00 a.m. to
prevent any bias in the concentration of saliva due Results
to circadian rhythm.[18] Children were also informed
not to eat or drink anything (except water) 1 h before The mean age of the participants was 5 (standard
saliva collection to minimize possible food debris and deviation [SD]: 0.69) years and the mean defs was 16.20
stimulation of saliva. (SD: 8.58). Green tea showed a statistically significant
fall in the colony counts of S. mutans and Lactobacilli
Microbial evaluation spp. (P < 0.001, P < 0.001); whereas only a numerical
The samples were transported to the microbiological fall in C. albicans colony count was found, which was
laboratory in box with ice-packs. They were tested not statistically significant (P = 0.264) [Table 1]. CHX
for the colony forming units (CFU) of S. mutans, showed a statistically significant fall in the colony
Lactobacilli spp., and C. albicans using mitis-salivarius- counts of S. mutans and Lactobacilli spp. (P < 0.001,
bacitracin agar, Rogosa agar, and HiCrome agar, P < 0.001); whereas a numerical increase was observed

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Thomas, et al.: Comparing anti-microbial efficacy of green tea and chlorhexidine mouth rinses on cariogenic microbes

in the colony count of C. albicans, which was also not against S. mutans and Lactobacilli spp. with some anti-
statistically significant (P = 0.264) [Table 1]. fungal activity against C. albicans. The antibacterial
effect of green tea mouth rinse is in accordance to the
On comparison against S. mutans, green tea mouth rinse previous reports where the authors stated that rinsing
was found to be significantly better than CHX mouth with green tea extract had valuable anti-cariogenic
rinse (P = 0.005). Against Lactobacilli spp., CHX mouth activities including inhibitory effect on cariogenic
rinse was found to be significantly more effective than bacteria by inhibiting the adherence of bacterial cells to
green tea mouth rinse (P < 0.001). Whereas against the tooth surfaces.[14,17,19,20] And also, green tea catechins
C. albicans, no statistically significant difference was maintain the salivary pH at a normal range, which is
found (P = 0.081) [Table 2]. not a favorable condition for cariogenic bacteria to
flourish.[21,22] The anti-fungal activity is in agreement to
Majority of the study participants in the green tea group the earlier in vitro studies that have reported that green
had a positive response to flavor (n = 10; 66.7%); smell tea polyphenols and catechins inhibit the growth of
(n = 8; 55.3%); and willingness to rinse (n = 10; 66.7%). C. albicans by 40% and 75%, respectively.[21,22]
However, the response of the study participants in
the CHX group was varying. Only 40% (n = 6) were CHX mouth rinse is one of the best proven
positive to flavor, 66.7% (n = 10) were positive to smell, commercially available mouth rinses due to its broad
and 46.7% (n = 7) were positive to willingness to rinse. spectrum anti-microbial activity and no systemic side
effects[23] Scientific studies have reported that CHX
mouth rinse was effective in reducing the colony
Discussion counts of S. mutans in plaque biofilm;[18,23,24] Lactobacilli
spp. colony counts,[23,25] and culturable microbes of
The purpose of this study was to evaluate and compare tongue and saliva.[24] The present study also confirmed
the anti-microbial efficacy of green tea mouth rinse the anti-bacterial activity of CHX mouth rinse against
to that of CHX mouth rinse on the level of salivary S. mutans and Lactobacilli spp. However, no anti-
S. mutans, Lactobacilli spp., and C. albicans in children. candida effect was found; in fact, a numerical increase
The present study was carried out under real life was observed which was not statistically significant.
conditions without altering the subjects’ routine oral This increase in C. albicans count could be assigned to
hygiene practices except that they were advised to use various factors such as a direct trigger of C. albicans by
a nonfluoridated dentifrice for cleaning their teeth. CHX or due to depression of the bacterial counts aiding
This was done to eliminate the bias resulting due in the proliferation of this opportunistic pathogen.
to additional anti-microbial effect of fluoride from Another probable explanation is the basic differences
the dentifrice. To standardize rinsing, all the study in outer cell structure between bacteria and fungi, with
participants were asked to rinse under monitoring at the latter having a rigid outer chitin wall making it
school. Thus, rinsing was carried out after breakfast less amenable for lysis.[26] Bioavailability of CHX may
and this could have also contributed toward reducing also have been compromised in this study because
the microbial challenge. rinsing was carried out only once daily and also, only
5 ml of mouth rinse was used, for reports have stated
According to the results of this study, green tea was it to be about 12 h with 10 ml of 0.2% of CHX mouth
found to be a very effective anti-bacterial mouth rinse rinse.[7,8] Most scientific reports have shown fungicidal

Table 1: Mean differential colony counts of Streptococcus mutans, Lactobacilli spp., and Candida albicans
in both the mouth rinse groups
Mouth rinse group n Microorganism Mean baseline (CFU/ml) Mean postrinse (CFU/ml) Mean difference (CFU/ml) Significance (P)
CHX 15 S. mutans 4.7×107 5.9×103 4.7×103 <0.001**
Lactobacilli spp. 5.2×105 3.9×105 1.3×105 <0.001**
C. albicans 1.7×105 3.03×105 −1.33×103 0.264
Green tea 15 S. mutans 2.9×108 1.9×108 1×108 <0.001**
Lactobacilli spp. 4.9×104 2.2×104 2.7×104 <0.001**
C. albicans 6.4×10 4
4.1×104
2.3×104 0.264
**Very highly significant (P < 0.001). S. mutans = Streptococcus mutans; C. albicans = Candida albicans; CFU/ml = Colony forming units per millimeter

Table 2: Inter comparison of the mean difference of colony counts of both the mouth rinse groups for
Streptococcus mutans, Lactobacilli spp., and Candida albicans
Dependent variable Group (I) Group (J) Mean difference (I–J) ×103 (CFU/ml) SE P
S. mutans CHX Green tea −208.412 59,868.01 0.005*
Lactobacilli spp. CHX Green tea 232.067 50.431 <0.001**
C. albicans CHX Green tea −176.333 72.176 0.081
*Significant (P = 0.005); **Very highly significant (P < 0.001). SE = Standard error; CHX = Chlorhexidine; S. mutans = Streptococcus mutans; C. albicans = Candida albicans

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Thomas, et al.: Comparing anti-microbial efficacy of green tea and chlorhexidine mouth rinses on cariogenic microbes

and fungi static action of CHX where it was used in 8. Ilango P, Arulpari M, Medona M, Abirami T. Chlorhexidine –
combination with other chemical solutions such as A miracle chemical. Int J Cur Res Rev 2013;5:26-34.
cetylpyridinium chloride, fluconazole, and chitosan 9. Niklaus P, Lindhe L, Lindhe J. Textbook of Clinical
due to their synergistic action.[27-29] Periodontology and Implant Dentistry. 5th ed. Blackwell
Publications 2008. p. 748-54.
On comparison of the anti-microbial efficacy of green 10. Kubo I, Murohi H, Himejima M. Antimicrobial activity of
tea mouth rinse to that of 0.2% CHX, we found that it was green tea flavor components and their combination effects.
significantly better against S. mutans, but less effective J Agric Food Chem 1992;40:245-8.
against Lactobacilli spp. and comparable against C. 11. Sakanaka S, Kim M, Taniguchi M, Yamamoto T.
albicans. There is a paucity of reports comparing the Antibacterial substances in Japanese green tea extract against
anti-bacterial effect of green tea and CHX mouth rinses Streptococcus mutans, a cariogenic bacterium. Agric Biol
whereas the anti-plaque property of these mouth rinses Chem 1989;53:2307-11.
has been reported to be similar.[19] While in another 12. Hamilton-Miller JM. Anti-cariogenic properties of tea
report, 0.5% green tea mouth rinse was reported to be (Camellia sinensis). J Med Microbiol 2001;50:299-302.
better than 0.2% CHX mouth rinse.[30] Kaur et al., have 13. Yoshihara A, Sakuma S, Kobayashi S, Miyazaki H.
suggested that green tea catechins helps to maintain Antimicrobial effect of fluoride mouthrinse on mutans
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condition for S. mutans to colonize and multiply.[19] 2001;23:113-7.
14. Si W, Gong J, Tsao R, Kalab M, Yang R, Yin Y. Bioassay-guided
In our study, the green tea mouth rinse was widely purification and identification of antimicrobial components in
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mouth rinse had poor acceptance rate and this is a 15. Awadalla HI, Ragab MH, Bassuoni MW, Fayed MT,
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16. American Academy of Pediatric Dentistry; American Academy
Conclusion of Pediatrics; American Academy of Pediatric Dentistry
Council on Clinical Affairs. Policy on early childhood
From the results of our study, it can be concluded caries (ECC): Classifications, consequences, and preventive
that green tea mouth rinse could be very good cost- strategies. Pediatr Dent 2005-2006;27 (7 Suppl):31-3.
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with long-term use of this mouth rinse. following green tea mouth rinse or sodium fluoride mouth
rinse use in children (randomized double-blind controlled
Financial support and sponsorship clinical trial). Dent Res J (Isfahan) 2011;8 (Suppl 1):S58-63.
Nil. 18. Pratten J, Wills K, Barnett P, Wilson M. In vitro studies of the
effect of antiseptic-containing mouthwashes on the formation
and viability of Streptococcus sanguis biofilms. J Appl
Conflicts of interest
Microbiol 1998;84:1149-55.
There are no conflicts of interest.
19. Kaur H, Jain S, Kaur A. Comparative evaluation of the
antiplaque effectiveness of green tea catechin mouthwash
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